Site-Specific Incorporation of Non-canonical Amino Acids by Amber Stop Codon Suppression in Escherichia coli

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

Download Full-text

In-Frame Amber Stop Codon Replacement Mutagenesis for the Directed Evolution of Proteins Containing Non-Canonical Amino Acids: Identification of Residues Open to Bio-Orthogonal Modification

PLoS ONE ◽

10.1371/journal.pone.0127504 ◽

2015 ◽

Vol 10 (5) ◽

pp. e0127504 ◽

Cited By ~ 8

Author(s):

James A. J. Arpino ◽

Amy J. Baldwin ◽

Adam R. McGarrity ◽

Eric M. Tippmann ◽

D. Dafydd Jones

Keyword(s):

Amino Acids ◽

Directed Evolution ◽

Stop Codon ◽

Amber Stop Codon ◽

Evolution Of Proteins

Download Full-text

IntI2 Integron Integrase in Tn7

Journal of Bacteriology ◽

10.1128/jb.184.6.1712-1721.2002 ◽

2002 ◽

Vol 184 (6) ◽

pp. 1712-1721 ◽

Cited By ~ 128

Author(s):

Karin Hansson ◽

Lars Sundström ◽

Alex Pelletier ◽

Paul H. Roy

Keyword(s):

Amino Acids ◽

Antibiotic Resistance ◽

Stop Codon ◽

Antibiotic Resistance Genes ◽

Peptide Sequence ◽

Integrase Gene ◽

Site Specific ◽

Class 1 Integrons ◽

Parental Allele ◽

Class 1

ABSTRACT Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.

Download Full-text